The present invention relates to a biosensor for rapid and simplified quantitation of a substrate in a sample with high accuracy.
Conventionally, methods using polarimetry, colorimetry, reductimetry, a variety of chromatography, etc. have been developed as the measure for quantitative analysis of sugars such as sucrose, glucose, etc. However, those conventional methods are all poorly specific to sugars and hence have poor accuracy. Among them, the polarimetry is simple in manipulation but is largely affected by the temperature at manipulation. Therefore, this method is not suitable for simple quantitation of sugar level at home by ordinary people.
On the other hand, a variety of biosensors have been developed recently which best utilize a specific catalytic action of enzymes.
In the following, a method of quantitative analysis of glucose will be described as an example of the method for quantitating a substrate in a sample solution. Conventionally known electrochemical quantitation of glucose is the method using a combination of glucose oxidase (EC 1.1.3.4; hereinafter abbreviated to "GOD") with an oxygen electrode or a hydrogen peroxide electrode (see "Biosensor" ed. by Shuichi Suzuki, Kodansha, for example).
GOD selectively oxidizes a substrate .beta.-D-glucose to D-glucono-.delta.-lactone using oxygen as an electron mediator. Oxygen is reduced to hydrogen peroxide during the oxidation reaction by GOD in the presence of oxygen. A decreased volume of oxygen is measured by the oxygen electrode or an increased volume of hydrogen peroxide is measured by the hydrogen peroxide electrode. The decreased volume of oxygen or, otherwise, the increased volume of hydrogen peroxide is proportional to the content of glucose in the sample solution. Therefore, glucose concentration in the sample solution can be quantitated based on the decreased volume of oxygen or the increased volume of hydrogen peroxide.
However, as speculated from the oxidation reaction by GOD, this prior art method has a drawback of great influences on the measurement result of the oxygen concentration in the sample solution. In the event that oxygen is absent in the sample solution, this method does not allow measurement itself.
Under the circumstance, a glucose sensor of new type has been developed which uses as the electron mediator an organic compound or a metal complex such as potassium ferricyanide, a ferrocene derivative, a quinone derivative, etc., in place of oxygen in the sample solution. This sensor reoxidizes a reduced form electron mediator resulting from the enzyme reaction in order to determine the glucose concentration in the sample solution based on an oxidation current produced by the reoxidation reaction. The use of such organic compound or metal complex as the electron mediator in place of oxygen secures precise placement of a known amount of GOD together with the electron mediator in their stable state on an electrode system in forming a reagent layer on the electrode system. At that time, the reagent layer may be integrated with the electrode system while keeping the former in almost dry state. Therefore, a disposable glucose sensor based on this technology has recently been noted considerably. Such disposable glucose sensor facilitates measurement of glucose concentration using a measurement device exclusive to the sensor by simple introduction of a sample solution into the sensor detachably connected to the measurement device. The application of this technique is not limited to glucose quantitation and may be extended to quantitation of any other substrate present in the sample solution.
Measurement using the above-mentioned sensor determines substrate concentration in a sample based on an oxidation current that flows upon oxidation of a reduced form electron mediator on a working electrode. However, when the sample is blood, fruit juice or something like that, any easy-to-be-oxidized substance, such as ascorbic acid, uric acid, etc., in the sample will be oxidized on the working electrode together with the reduced form electron mediator. This oxidation of easy-to-be-oxidized substance sometimes produces an adverse effect on the measurement result.
The measurement using the above-mentioned sensor has another problem of simultaneous developments of reduction of the electron mediator carried on the reagent layer with generation of hydrogen peroxide by using dissolved oxygen in the sample as an electron mediator. Furthermore, the hydrogen peroxide thus generated acts to reoxidize the reduced form electron mediator. This sometimes results in production of negative errors in the results of measurement by the dissolved oxygen when the substrate concentration is to be determined based on the oxidation current of the reduced form electron mediator.